1. Field of the Application
The present application relates to inhibitors of extracellular signal-regulated kinase (ERK), compositions containing and methods for making same, and their use.
2. Discussion of the Background
Over the past decade, the inhibition of protein kinases has emerged as one of the most promising therapeutic approaches for treating cancer. Significant efforts in developing anti-cancer therapies have focused on the targeted inhibition of the extracellular signal-regulated kinase (ERK) pathway in cancers that contain activating mutations in upstream RTK, Ras and BRaf proteins. The usefulness of many of these targeted compounds has been limited, however, because of toxicity, lack of efficacy, and development of drug resistance through mutations or activation of alternate survival pathways. The identification of an activating point mutation (primarily V600E) in the BRaf kinase in melanoma patients led to the discovery of PLX4032 (vemurafenib), a drug which selectively inhibits the mutated and constitutively active form of BRaf. While patients showed remarkable initial responses to PLX4032, they invariably developed a resistance to the drug and consequently poor survival outcomes. A major contributor to PLX4032 drug resistance and poor prognosis is the re-activation of the extracellular signal-regulated kinases (ERK). Currently, all small molecule kinase inhibitors in the clinic act by blocking ATP binding or catalytic sites and inhibit all enzyme activity. While this approach may be useful in some cancers, the inevitable drug resistance that occurs in melanoma remains a barrier to achieving sustained therapeutic responses.
The mitogen activated protein kinase (MAPK) family consist of four major members including the extracellular signal-regulated kinases-1 and 2 (ERK1/2), c-Jun N-terminal kinases (JNK), p38 MAP kinases, and ERK5. The MAPK proteins are serine/threonine kinases involved in signal transduction pathways that regulate cell proliferation, apoptosis, differentiation, migration, and inflammation responses to a variety of extracellular signals. MAPK proteins regulate cellular functions through phosphorylation of a diverse number of substrates. In particular, the ERK1/2 proteins have been implicated in the phosphorylation of well over 100 substrates and stringent control over interactions of ERK1/2 with substrate proteins that allow efficient phosphate transfer is essential for proper cellular function. Unregulated activation of the ERK1/2 pathway is often observed in a variety of cancers, which contributes to uncontrolled cell proliferation, survival, and resistance to anti-cancer drugs. Although several selective ATP-competitive inhibitors of ERK1/2 have been developed, these compounds have not advanced to the clinic and do not allow examination of select ERK functions that are dependent on interactions with specific substrate proteins.
Many ERK1/2 substrate and interacting proteins, including the Elk-1 transcription factor, p90 ribosomal S6 kinase-1 (RSK-1), the caspase-9 protease, and the HePTP protein tyrosine phosphatase, contain a DEJL (docking site for ERK and JNK, Leu-X-Leu) motif or D-domain that is involved in kinase recognition. The D-domain consists of basic residues followed by a hydrophobic Leu-X-Leu motif, and interacts with acidic and hydrophobic regions in the carboxy terminus of ERK1/2, referred to as the common docking (CD domain) or D-recruitment site (DRS). A second docking domain, known as the F-site or DEF (docking site for ERK, Phe-X-Phe) motif, has been identified on several ERK1/2 substrates, including transcription factors like Elk-1 and c-Fos, A-Raf kinase, the kinase suppressor of Ras-1 (KSR-1) scaffold protein, and nuclear pore proteins like NUP153 and NUP214. The F-site is typically separated from the phosphorylation site by 6-10 amino acids, whereas the D-domain may be located 20 amino acids further from the phosphorylation site to accommodate the spatially separated hydrophobic interactions.
The DRS on ERK2 includes residues Asp316 and Asp319, adjacent hydrophobic amino acids, and ED domain residues (Glu160/Asp161 for p38α MAP kinase and Thr157/Thr158 for ERK2) that facilitate selective interactions between D-domain containing substrates and MAP kinases. F-site containing substrates interact with hydrophobic regions that make up the F-recruitment site (FRS) on ERK2 and include residues Leu198, Tyr231, Leu232, Leu235, and Tyr261. Other MAP kinases, including p38α, may also utilize a FRS-like binding motif during substrate recognition. Beyond the DRS and FRS sites, experimental studies using ERK2 mutants have implicated other residues that may be important for ERK interactions with substrates or other regulatory proteins. Indeed, ERK2, and other MAP kinases, have a unique insert in the kinase homology region that may regulate it's interactions with upstream activating MEK proteins. Given the large number of substrates regulated by ERK1/2 proteins, it is likely that additional docking sites will be identified on ERK1/2 proteins that regulate specific protein-protein interactions.
Promising new drugs that target the mutated and active form of BRaf in melanoma cells have clinical limitations due to the unanticipated activation of the ERK1/2 pathway through alternate mechanisms and the activation of compensatory signaling pathways that lead to drug resistance. In addition, MEK-independent mechanisms may activate ERK in cancer cells resistant to clinically relevant Raf or MEK inhibitors. A recent study suggested that targeting ERK directly with ATP competitive inhibitors may overcome resistance to MEK inhibitors. Whether complete blockade of ERK using this approach will induce resistance pathways as observed with the MEK1/2 and BRaf inhibitors remains to be determined.
Regulated activity of ERK1/2 serves integral roles in normal cell processes, whereas unregulated and constitutively active ERK1/2 sustains cancer cell proliferation and survival. Thus, in the absence of discriminating between cancer and normal cells, inhibitors that completely block ERK1/2 signaling are destined to have toxicity to normal cells. Despite extensive data demonstrating the role of ERK1/2 signaling in normal cellular processes and disease states, there is surprisingly little known about the mechanisms involved in ERK1/2 recognition and binding interactions with substrate proteins. Given that ERK proteins may have nearly 300 interacting partners, with over half of these being phosphorylated substrates, there is a knowledge gap regarding ERK1/2 interactions with substrates that are especially relevant to cancer cell proliferation and survival. The identification of molecules that selectively disrupt these interactions will have a significant impact on the development of novel inhibitors that prevent the phosphorylation and regulation of ERK substrates involved in disease processes while preserving ERK functions in normal cells.
Although the ERK1/2 proteins are key drivers of proliferation and survival in many cancers, they also play essential roles in regulating normal cellular processes. Competitive ATP inhibitors, such as conventionally used in the treatment of cancer, may have toxic effects due to off-target effects involving other kinases. There exists selective pressure on cancer cells to induce compensatory pathways that results from complete ablation of ERK1/2 signaling. Currently, chemotherapeutic drugs that block ERK-regulated pathways by targeting MEK1/2 and BRaf proteins invariably cause drug resistance due to treatment-induced activation of compensatory survival proteins and MEK1 mutations.